1. Field of the Invention
The present invention relates generally to the field of molecular biology, and in particular, methods and compositions for the detection and treatment of diabetes, neoplasms, hyperinsulinemia, and obesity. More particularly, certain embodiments concern the DNA segments encoding novel murine- and human-derived polypeptides comprising agouti and agouti-related proteins. In certain examples, the invention concerns the use of these nucleic acids to regulate fatty acid metabolism and treat various forms of cancer, including tumors. Methods, compositions, kits and devices are also provided for identifying compounds which are inhibitors of agouti activity in vitro and in vivo.
2. Description of Related Art
The agouti locus (a) in chromosome 2 regulates the differential production of black and yellow pigment granules that give rise the agouti coat color of the mouse. Agouti coloration, which is the true wild-type coat color of mice, is unusual in that it arises not from a homogenous pigmentation of the pelage, but rather from a banded coloration pattern in which each hair is black with a subapical band of yellow. One of the most interesting aspects of the agouti locus is that it functions within the microenvironment of the hair follicle (Silver and Russell, 1955; Silvers, 1958a, 1958b, Silvers, 1961, Silvers, 1979), unlike may other coat color genes, which act in a cell-autonomous manner within the melanocytes. Therefore, agouti must be regulating coat pigmentation by some direct or indirect form of intercellular signaling within the follicular environment.
Like many other genes that play a role in the regulation of coat pigmentation in the mouse, the agouti locus contributes to essential developmental processes unrelated to pigmentation (Geissler et al., 1988; Witte, 1990; Epstein et al., 1991; Mercer et al., 1991). For example, some of the individual alleles at the agouti locus are associated with embryonic lethality, obesity, diabetes, and the development of tumors in a wide variety of tissues. In fact, the lethal yellow (A.sup.y) mutation at agouti was the first embryonic lethal mutation to be characterized in the mouse (Cuenot, 1905). Embryos homozygous for A.sup.y die very early in development, around the time of implantation, possibly owing to a defect in trophectoderm differentiation (Eaton and Green, 1963; Calarco and Pederson, 1976; Papaioannou and Gardner, 1979).
Genetic analyses of numerous a locus mutants have been ongoing for nearly a century, and have led to the identification of at least 18 dominant and recessive alleles and pseudoalleles of agouti (Silvers, 1979; Green, 1989). Different combinations of alleles account for an array of different phenotypes, ranging from subtle differences in coat color as compared with the wild type, to drastic changes in the distribution of pigmentation in different regions of the animal, particularly across the dorso-ventral surface. An intricate dominance hierarchy exists in which alleles associated with phaeomelanin (yellow) production are generally dominant over alleles associated with eumelanin (Black or brown, depending on alleles at other loci) production. This relationship is exemplified by several alleles that date back to the mouse fancy: lethal yellow (A.sup.y), which confers an all-yellow phenotype in the heterozygous condition, black-and-tan(a.sup.t), which gives rise to an all-black dorsum and an all-yellow ventrum (Dunn, 1928), nonagouti (a), which gives rise to a predominantly black phenotype, except for small amounts of phaeomelanin around the pinnae, nipples, and perineum, and extreme nonagouti (a.sup.c), which confers a completely black phenotype (Hollander and Gowen, 1956).
The large number of alleles and the wide range of phenotypes associated with the agouti locus have been used as evidence by some investigators to propose that the agouti locus is comprised of multiple "mini-loci" and not a single gene. According to this hypothesis, each gene of the mini-locus plays a role in regulating pigmentation in different parts of the body, particularly over the dorsal and ventral surfaces, and around the pinnae, nipples, and perineum. Support for this assertion stems from the finding that changes from yellow to black pigmentation proceed from the dorsal to the ventral regions as one progresses from the most dominant to the most recessive mutation of the agouti allelic series. For example, phaeomelanin progressively disappears from the mid-dorsum with A.sup.i /a (A.sup.i, intermediate yellow), from the lateral dorsum with a.sup.t /a.sup.t, from the ventral surface with a/a, and from the pinnae, nipples, and perineum with a.sup.c. With the mini-locus hypothesis, different genes should be affected by mutations associated with the individual alleles in the hierarchy. The present invention demonstrates that the structure and expression of the same gene is affected by mutations at the top (A.sup.y), middle (a.sup.t and a), and bottom (a.sup.c) of the allelic series. These results disprove the mini-locus hypothesis.
Although the agouti alleles have been extensively characterized with classical genetic techniques, the structure of the gene(s) responsible for a locus function had not been determined until the present invention. Attempts by others to isolate the gene using positional cloning techniques, failed to isolate the gene (Barsh and Epstein, 1989a; Siracusa et al., 1987a; Siracusa et al., 1989; Siracusa, 1991).
A radiation-induced inversion mutation, called Is(17;In2)Id,aJGso (abbreviated IslGso), which contains DNA breakpoints in the limb deformity (Id) and agouti loci, two regions that are normally separated by 22 cM on chromosome 2 (Woychik et al., 1990a; Bultman et al., 1991) was previously described. Utilizing a DNA probe from the Id.sup.Hd insertional mutant (Woychik et al., 1985), 22 cM were jumped with the inversion which allowed a region of DNA that maps to the agouti locus to be identified (Woychik et al., 1990a). Moreover, this region also hybridizes to sequences that are rearranged in several agent-induced a locus mutations (Bultman et al., 1991).